Tylosin Residue in Chicken: Detection with ELISA, Four Plate Test, HPLC, Effect of Heat Treatment and implications for Human Health

Document Type : Research Article

Authors

• Innocent Nwankwo ¹
• Stella Onwumere-Idolor ²
• Ekene Ezenduka ¹
• John Nwanta ¹
• Aruh Anaga ³

¹ Department of Veterinary Public Health and Preventive Medicine, University of Nigeria, Nsukka, Nigeria.

² Department of Veterinary Public Health and Preventive Medicine, University of Nigeria, Nsukka & Department of Animal Health and Production Technology, Delta state Polytechnic, Ozoro, Delta State, Nigeria.

³ Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Nigeria.

10.22067/ijvst.2025.88457.1389

Abstract

Tylosin residues (TR) in chicken meat pose potential health risks to consumers. This study aimed to detect and quantify TR in chicken tissues from Ikpa slaughterhouse, Nsukka and evaluate the effect of heat treatment on TR concentrations. Sixty randomly sampled chicken were processed, and their muscle, liver, and kidney tissues were collected and tested for TR at raw and after ten, fifteen , and twenty minutes of cooking and microwaving using enzyme-linked immunosorbent assay (ELISA), four plate test (FPT), and high-performance liquid chromatography (HPLC). Of the 180 tissues, 93 (51.7%) were positive for TR. The prevalence of TR was 40% in muscles, 55% in liver, and 60% were in kidney samples with ELISA. Six liver samples exceeded the maximum residue level (MRL) of100 µg/kg. Cooking and microwaving reduced TR concentrations by 97-100% in muscle and liver tissues using HPLC. The mean inhibition zones decreased by 87-100% after cooking and microwaving using FPT. Chicken at Ikpa slaughterhouse, Nsukka have TR even in concentrations above the MRL but were significantly eliminated (p ˂ 0.05) after 20 minutes of heat treatments (cooking or microwaving). Hence, mitigating the health risks associated with TR in meat requires regular screening and quantification, public awareness campaigns targeting consumers of raw or improperly cooked chicken, strict policies on antibiotics use in poultry, and enhanced meat handling and processing practices in food industry.

Keywords

• Tylosin Residue Analysis
• Chicken
• Cooking
• Microwaving
• ELISA
• Four Plate Test
• HPLC
• Food Safety

Main Subjects

• Food hygiene

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References

1.    EuroStat. Agricultural Production—Livestock and Meat. Accessed December 15, 2022.https://ec.europa.eu/eurostat/statisticsexplained/index.php?title=Agricultural production livestock and meat#Poultry. 
2.    USDA. United States Department of Agriculture Foreign Agricultural Service October 12, 2023 Livestock and Poultry: World Markets and Trade. Accessed November 10, 2023. https://public.govdelivery.com/accounts/USDAFAS/subscriber/new.
3.    F.A.O. Gateway to Poultry Production and Products. Accessed March 15, 2023. https://www.fao.org/poultry-production-products/production/en/. 
4.    Njoga EO, Ogugua AJ, Nwankwo IO, Awoyomi OJ, Okoli CE, Maigawu BD. Oyeleye FA, et al. Antimicrobial drug usage pattern in poultry farms in Nigeria: implications for food safety, public health, and poultry disease management. Vet Italiana. 2021;57(1):5-12. Doi: 10.12834/VetIt.2117.11956.1.
5.    Patel T, Marmulak T, Gehring R, Pitesky M, Clapham MO, Tell LA. Drug residues in poultry meat: A literature review of commonly used veterinary antibacterials and anthelmintics used in poultry. Journal of Veterinary Pharmacology and Therapy. 2018; 41(6):761-789. Doi: 10.1111/jvp.12700.
6.    Kim MJ, Kim JY, Choi JD, Moon G. Re-evaluation of a microbiological acceptable daily intake for tylosin based on its impact on human intestinal microflora. Toxicology Research. 2023;1-6. Doi: 10.1007/s43188-023-00179-z.
7.    Soliman AM, Sedeik M.  Pharmacokinetics and tissue residues of tylosin in broiler chickens. Pharmacology and Pharmacy. 2016;7(1):36-42. Doi: 10.4236/pp.2016.71006.
8.    De Wasch K, Okerman L, Croubels S, de Brabander H, van Hoof J, De Backer P. Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory test. Analyst. 1998;123(12):2737-41. Doi: 10.1039/a804909b.
9.    Javadi A, Mirzaie H, Khatibi SA. Effect of roasting, boiling and microwaving cooking methods on Enrofloxacin residues in edible tissues of broiler. African Journal of Pharmacy and. Pharmacology. 2011;5(2):214-218. Doi: 10.5897/AJPP10.378
10.    Salaramoli J, Heshmati A, Kamkar A, Hassan J. Effect of cooking procedures on tylosin residues in chicken meatball. Journal für Verbraucherschutz und Lebensmittelsicherheit  2015;11(1):53-60. Doi: 10.1007/s00003-015-1005-1. 
11.    Gowtham P, Muthukumar M, Eswara RB, Kalpana S, Keerthika V, Dani N. Impact of cooking methods of meat on antibiotic residues: Review. TPI International  Journal of science and technology. 2020; 9(12):303-9.Doi: 10.22271/tpi.2020.v9.i12e.5454.
12.    El Tahir Y, Elshafie EI, Asi MN, Al-Kharousi K, Al Toobi AG, Al-Wahaibi Y, Al-Marzooqi W. Detection of Residual Antibiotics and Their Differential Distribution in Broiler Chicken Tissues Using Enzyme-Linked Immunosorbent Assay. Antibiotics (Basel). 2021;10(11):1305. Doi: 10.3390/antibiotics10111305. 
13.    Debeb D. Review on Effect of Antimicrobial Residual on Human. Journal of Veterinary Science and Medical Diagnostics. 2021;10(7):1-4. 
14.    Prats C, Francesch R, Arboix M, Pérez B. Determination of tylosin residues in different animal tissues by high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;766(1):57-65. Doi: 10.1016/s0021-9673(01)01325-5. 
15.    Ghanjaoui ME, Mandil A, Ait Sidi Mou A, Slimani R. High-performance liquid chromatography quality control. International Journal of Advances in Chemistry. 2020;8:160-169. Doi: 10.14419/ijac.v8i1.30723
16.    Treiber FM, Beranek-Knauer H. Antimicrobial Residues in Food from Animal Origin-A Review of the Literature Focusing on Products Collected in Stores and Markets Worldwide. Antibiotics (Basel). 2021 May 6;10(5):534. doi: 10.3390/antibiotics10050534.
17.    Jayalakshmi K, Paramasivam M, Sasikala M, Tamilam TV, Sumithra A.  Review on antibiotic residues in animal products and its impact on environments and human health. Journal of Entomology and Zoological Studies 2017;5(3):1446-1451.  Doi:10.47672/ejah.1104
18.    Tasci F, Secilmis Canbay H, Doganturk M. Determination of antibiotics and their metabolites in milk by liquid chromatography-tandem mass spectrometry method. Food Control. 2021;127:108-147. Doi: 10.1016/j.foodcont.2021.108147.
19.    Khaniki GJ, Aghaee EM, Sadighara P. Chemicals and drugs residue in meat and meat products and human health concerns. Journal of Food Safety and Hygiene 2018;4:1-7. 
20.    Nwankwo IO, Ezenduka EV, Nwanta JA, Ogugua AJ, Audu BJ. Prevalence of Campylobacter spp. and antibiotics resistant E. coli on poultry carcasses and handlers’ hands at Ikpa slaughter, Nsukka, Nigeria. Notulae Sciencia Biologica. 2021;13:(2)10866. Doi:10.15835/nsb13210866.
21.    Onwumere-Idolor OS, Nwankwo IO, Ezenduka EV, Nwanta JA and Anaga AO. Comparison of Premi® test, Four Plate Test, ELISA, and HPLC tests in the detection of tylosin residue and its depletion time in chickens treated with tylosin by intramuscular injection and oral routes. Journal of Veterinary and Applied Sciences. 2024;14(1):469–480. 
22.    Onwumere-Idolor OS,  Ogugua A J,  Ezenduka EV, Nwanta JA, Anaga A. Prevalence of Antimicrobial Residues in Tissues of Broilers Sold at a Local Market in Enugu State, Nigeria Using the European Four Plate Test. Journal of  Food Nutrition and Research. 2022;  Doi: 10.26502/jfsnr.2642-110000102. 
23.    International Programme on Chemical Safety & Joint FAO/WHO Expert Committee on Food Additives. Toxicological evaluation of certain veterinary drug residues in food /prepared by the seventieth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). World Health Organization. 2009:https://apps.who.int/iris/bitstream/handle/10665/44086/9789241660617_eng.pdf?sequence=1&isAllowed=y  Accessed on 15 September 2022. 
24.    Murray SA, Holbert AC, Norman KN, Lawhon SD, Sawyer JE, Scott HM. Effects of Tylosin, a Direct-Fed Microbial and Feedlot Pen Environment on Phenotypic Resistance among Enterococci Isolated from Beef Cattle Feces. Antibiotics. 2022;11(1):106. Doi: 10.3390/antibiotics11010106.   
25.    Cazer CL, Eldermire ERB, Lhermie G, Murray SA, Scott HM, Gröhn YT. The effect of tylosin on antimicrobial resistance in beef cattle enteric bacteria: A systematic review and meta-analysis. Preventive Veterinary Medicine. 2020;176:104934. Doi: 10.1016/j.prevetmed.2020.104934.  
26.    WHO Antibiotics resistance: Accessed October 15, 2022. https://www.who.int/news-room/fact- sheets/detail/antibiotic-resistance.  
27.    Ali A, Ahmad N, Liaqat A, Farooq MA, Ahsan S, Chughtai MFJ, et al. Safety and quality assessment of street-vended barbecue chicken samples from Faisalabad, Pakistan. Food Science and Nutrition. 2022;11(2):930-939. Doi: 10.1002/fsn3.3127.  
28.    Codex Alimentarius Commission Maximum. Residue Limits for Veterinary Drugs in Foods Updated as at the 34th Session of the Codex Alimentarius Commission 2011 Accessed  March 14, 2023.  
29.    Badger-Emeka LI, Emeka PM, Okosi M. Evaluation of the extent and reasons for increased non-prescription antibiotics use in a University town, Nsukka Nigeria. International  Journal of Health Sciences. (Qassim). 2018;12(4):11-17.  
30.    Chah JM, Nwankwo SC, Uddin IO, Chah KF. Knowledge and practices regarding antibiotic use among small-scale poultry farmers in Enugu State, Nigeria. Heliyon. 2022: 26;8(4):e09342. Doi: 10.1016/j.heliyon.2022.e09342.  
31.    Kolanovic BS, Bilandzic N, Varenina I, Bozic D. Tylosin content in meat and honey samples over a two-year perıod in Croatia. Journal of Immunoassay Immunochemistry. 2014; 35: 37-47. Doi: 10.1080/15321819.2013.784198.
32.    Pavlov AI, Lashev LI, Vachin, Rusea V. Residues of antimicrobial drugs in chicken meat and offals. Trakia Journal of Sciences 2008;6(1):23-25.  
33.    JECFA. Evaluation of Certain Veterinary Drug Residues in Animals and Foods (Sixty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO              2006: Technical Report Series, No. 939.  
34.    Gürel Yücel M, Seçilmiş H, Taşçı F. Investigation of tylosin and tilmicosin residues in meat by high-performance liquid chromatography method. Polish Journal of Veterinary Sciences. 2023; 26( 1):39–46. Doi: 10.24425/pjvs.2023.145005.
35.    Tao Y, Yu G, Chen D, Pan Y, Liu Z, Wei H, Peng D, Huang L, Wang Y, Yuan Z. Determination of 17 macrolide antibiotics and avermectins residues in meat with accelerated solvent extraction by liquid chromatography-tandem mass spectrometry. Journal of Chromatography B Analytical Technology and Biomedical Life Sciences. 2012;897:64-71. Doi: 10.1016/j.jchromb.2012.04.011.
36.    Arslanbaş E, Şahin S, Kalın R, Moğulkoç MN, Güngör H. Determination of some antibiotic residues by HPLC method in chicken meats prepared for consumption. Erciyes University Veteriner Fakultasi Dergisi. 2018;15:247-252. Doi: 10.32707/ercivet.477315.
37.    Heshmati A, Kamkar A, Salaramoli J, Hassan J, Jahed GH. The effect of two methods cooking of boiling and microwave on tylosin residue in chicken meat. Iranian Journal of  Nutritional Science and Food Technology. 2013;8(1):231-240.  
38.    Pikkemaat MG. Microbial screening methods for detection of antibiotic residues in slaughter animals.  Analytical and Bioanalytical Chemistry. 2009;395(4):893–905. Doi: 10.1007/s00216-009-2841-6. 
39.    Shaltout AF, Mohammed A, El S, Heba MF. Studies on antibiotic residues in beef, and effect of cooking and freezing on antibiotic residues beef samples. Scholarly Journal of food nutrition. 2019; 2(1).Doi: 10.32474/DSJFN.2019.02.000129.